1.Field of the Invention
This invention relates to cell lines developed from differentiated tissue of insects, including midgut tissue; particularly, to cell lines which are susceptible to baculoviruses and may be used to replicate such viruses.
Abbreviations
Abbreviations or definitions used in the disclosure are as follows: AcMNPV, Autographa californica multiply-enveloped nuclear polyhedrosis virus; TnSNPV, Trichoplusia ni singly-enveloped nuclear polyhedrosis virus; MOI, multiplicity of infection; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; NPV, nuclear polyhedrosis virus.
2. Description of the Related Art
Insect cell culture has been used broadly in insect virology research since the first insect cell line was established in 1962 (Grace, T.D.C., Establishment of four strains of cells from insect tissue grown in vitro, Nature, 195:788-789). The general use of tissue cell lines for the culture or replication of pathogenic microorganisms is well established, and production of viral insecticides in cell culture has many advantages over their cultivation in vivo ((Weiss, S.A. and J.L. Vaughn, Cell culture methods for large-scale propagation of baculovirus, in "The Biology of Baculoviruses," vol. II "Practical Application for Insect Control" (Granados and Federici, eds., CRC Press, Boca Raton, FL), pp. 63-87 (1986)) and (Granados et al., Production on viral agents in invertebrate cell cultures, in "Biotechnology in Invertebrate Pathology and Cell Culture" (Maranorosch, K., ed., Academic Press, San Diego/N.Y.), pp. 167-181, (1987)). However, specific microorganisms cannot be cultured in all cell lines, even in all cell lines from the same order. Vaughn, in Invertebrate Tissue Culture, Research Applications (Academic Press, N.Y./London, 1976, pp. 295-303) discusses the development of insect cell lines and notes that, for example, cell lines from Heliothis zea are not capable of complete replication of the nuclear polyhedrosis virus obtained from Heliothis zea itself. It should be apparent from the above that the ability of a given cell line to replicate a given microorganism cannot be predicted from results in cell lines of different species or in different lines from the same species.
In recent years, baculovirus expression vectors have been widely used as vectors for foreign gene expression in insect cells (Luckow, V.A. and M.D. Summers, High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors, Virology, 170:311-39 (1988)). Therefore, insect cell culture has become more important as a potentially attractive system for producing viral insecticides and expressing foreign gene products of interest in the areas of biology, medicine, and agriculture.
In addition, Bacillus thuringiensis, a gram-positive soil bacterium, has been found to produce crystalline inclusions during sporulation, which consists of insecticidal proteins exhibiting a highly specific insecticidal activity (Aronson, et al., Bacillus thuringiensis and related insect pathogens, Microbiol. Rev. 50:1-24 (1986)), and many B. thuringiensis strains with different insect hosts have been identified (Burges, H.D., ed., Microbial Control of Pests and Plant Diseases 1970-1980, Academic Press, Inc., London (1981)). The cloning of these insecticidal crystal protein genes and their expression in plant-associated microorganisms or transgenic plants has provided potentially powerful alternative strategies for the protection of crops against insect damage (Hofte and Whiteley, Insecticidal crystal proteins of Bacillus thuringiensis, Microbiol. Rev. 53:242 (1989)). These crystal proteins act on the midgut cells of the insects, binding to specific midgut cell membrane receptors (Hofmann et al., Specificity of Bacillus thuringiensis .delta.-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts, Proc. Natl. Acad. Sc. 85:7844-7848 (1988)), causing the said midgut cells to swell and burst, and the insect stops eating and dies (Hofte and Whiteley, p. 242 (1989)).
Prior to this invention, no cell line had been established from midgut tissue of any insect species, and this hampered studies of the mode of action of these crystal proteins (Hofte and Whiteley, pp. 242-255 (1989)).
Cell lines have been established from other types of tissue from several species of the order Lepidoptera, which includes some of the most significant agricultural pests. Lynn et al. reported lines from embryos and fat body tissue of Lymantria dispar, and from the testes of Heliothis virescens, in 1988. Mitsuhashi established a cell line from fat body tissue of Leucania separata in 1983. Hink established the first such line from Trichoplusia ni, the cabbage looper, in 1970; it was derived from ovarian tissue.
Up to the time of this invention, a midgut cell line of the cabbage looper had not been established. Harrap and Robertson, as early as 1968, indicated that nuclear polyhedrosis viruses (NPVs) may infect a larger percentage of midgut cells in vivo, than fat body cells in vivo (Harrap, K.A. and J.S. Robertson, A possible infection pathway in the development of a nuclear polyhedrosis virus, J. gen Virol. 3:221-225 (1968)), making a midgut cell line, which may be used to reproduce baculoviruses, even more attractive.
Cell lines from Trichoplusia ni eggs have been established and infected ((Rochford et al., Establishment of a cell line from embryos of the Cabbage Looper, Trichoplusia Ni (Hubner), In Vitro 20: 823-825 (1984)) and (Granados et al., Replication of the Trichoplusia ni Granulosis and Nuclear Polyhedrosis Viruses in cell cultures, Virology 152: 472-476 (1986))), however, up to the time of this invention, a Trichoplusia ni embryonic cell line which is highly susceptible to numerous baculoviruses and efficiently supports replication of baculoviruses had not been established. Rochford et al. (1984) developed a Trichoplusia ni egg cell line (IPLB-TN-R) that is susceptible to only one of six baculoviruses tested, the Autographa californica multiply-enveloped nuclear polyhedrosis virus (AcMNPV). In addition, AcMNPV polyhedra production in the IPLB-TN-R cell line occurs later than desirable (beginning at 18 and 39 hours post infection), indicating an inefficient baculovirus replicating cell line. The BTI-TN-5B1-28 embryonic cell line reported by Granados et al. (1986) is moderately susceptible to infection by AcMNPV and Trichoolsia ni singly-enveloped nuclear polyhedrosis virus (TnSNPV).